Salmonella is a genus of bacteria that are causative of severe infections, notably of food or beverage toxi-infections, leading to bacterial enteric illness in both humans and animals, more particularly to salmonellosis, which include gastro-enteritis, as well as typhoid and para-typhoid fevers.
S. enterica subsp. enterica serovar Typhi and some S. enterica subsp. enterica serovar Paratyphi strains are the causative agents of typhoid fever.
There are currently more than 2,500 Salmonella serovars. Millions of human cases are reported worldwide every year, and the diseases result in thousands of deaths.
In recent years, problems related to Salmonella have increased significantly, both in terms of incidence and severity of cases of human salmonellosis.
Prior art method for the detection of Salmonella in food products comprises microbiology methods, which are described in European Standard ISO EN 6579:2002. Such food testing methods comprise lengthy culture steps, namely:                i. a pre-enrichment step on a non-selective liquid medium, which comprises the inoculation of a sample of the material to be assayed for, in buffered peptone water, and the incubation of the inoculated medium at 37° C. for 18±2 hours,        ii. a selective pre-enrichment step, which is performed on two selective liquid media, by:                    a. inoculating 0.1 mL of the incubated medium in 10 mL of RVS broth (Rappaport Vassiliadis single component enrichment broth), and incubating at 41.5±1° C. for 24±3 hours, and            b. inoculating 1 mL of the incubated medium in 10 mL of MKKTTn broth (Muller Kauffman Tetrathionate-novobiocine broth), and incubating at 37±1° C. for 24±3 hours,                        iii. an isolation step, which comprises inoculating an aliquot of each of the two above-mentioned liquid cultures, in two selective solid media, namely one XLD medium and another medium enabling the growth of lactose-positive Salmonella strains, and incubating these solid media at 37±1° C. for 24±3 hours,        iv. an identification step, and        v. a confirmation step, which comprises an agar culture at 37±1° C. for 24±3 hours, as well as a biochemical and a serological confirmation.        
This method is the Standard International ISO method for the detection of Salmonella. 
Other methods have been developed, which uses molecular biology, more particularly nucleic acid hybridization.
Illustrative of such methods are the method described in EP 0 721 989 B1 in the names of INSTITUT PASTEUR and INSERM (U.S. Pat. No. 5,824,795; U.S. Pat. No. 6,080,545), wherein oligonucleotides are disclosed to be useful as primers and probes. More particularly, oligonucleotides lag3 and lag6 are disclosed, wherein lag6 can be used as a primer within a primer pair formed with another oligonucleotide, namely lag5, and wherein lag3 can be used as a revealing probe.
The present application relates to an improved set of primers and probe, and to an improved method of Salmonella detection, which do not have the drawbacks of prior art techniques, and which further show unexpected effects and advantages.
The present application notably provides a set of primers and a probe, which can be used in multiplex in the same tube in real-time amplification, and which thereby enables to cover seven Salmonella groups in a single-tube operation. The set of primers and probe of the invention has the further advantage of having an increased sensitivity, without any loss in specificity.